Biosensing with Fluorescent Carbon Nanotubes

Abstract Biosensors are powerful tools for modern basic research and biomedical diagnostics. Their development requires substantial input from the chemical sciences. Sensors or probes with an optical readout, such as fluorescence, offer rapid, minimally invasive sensing of analytes with high spatial and temporal resolution. The near‐infrared (NIR) region is beneficial because of the reduced background and scattering of biological samples (tissue transparency window) in this range. In this context, single‐walled carbon nanotubes (SWCNTs) have emerged as versatile NIR fluorescent building blocks for biosensors. Here, we provide an overview of advances in SWCNT‐based NIR fluorescent molecular sensors. We focus on chemical design strategies for diverse analytes and summarize insights into the photophysics and molecular recognition. Furthermore, different application areas are discussed—from chemical imaging of cellular systems and diagnostics to in vivo applications and perspectives for the future.


Introduction
Future challenges in medicine such as early disease detection, point-of-care diagnostics,a nd tailored therapies require novel methods of biosensing. Additionally,biosensors can provide insights into the complex dynamics of biological and chemical systems.C onsequently,t hey are essential tools for both fundamental research and biomedicine.Inparticular, optical sensing approaches possess ag reat potential for contactless real-time readouts that are required in biomedical research, as well as industrial healthcare and agriculture applications. [1][2][3] During the last decade,the field of biosensors based on nanomaterials has seen vast improvements. [4,5] These materials include carbon-based nanomaterials such as graphene,g raphene quantum dots,a nd carbon nanotubes (CNTs). [4,[6][7][8] Here,s ingle-walled carbon nanotubes (SWCNTs) are of particular interest. Their optoelectronic properties are sensitive to the surrounding environment, which makes them suitable for highly selective biosensing. [8][9][10][11][12][13][14][15][16] When dispersed in aqueous solutions,S WCNTs fluoresce without bleaching in the near-infrared region (NIR, around l = 870-2400 nm). [17,18] This region of the electromagnetic spectrum is beneficial for detection and imaging as it offers an ultralow background and high penetration depths in biological tissues (tissue transparencyw indow). [1,2,9,[19][20][21] Fluorescence methods using common visible fluorophores often suffer from high scattering,absorption, and autofluorescence, which limits the penetration depth and signal to noise ratios. [1] Additionally,p hototoxicity is increased by excitation of common fluorophores with visible (Vis) or ultraviolet (UV) light. Consequently,S WCNTs offer an advantage as they combine the biocompatibility and photostability required for optical sensing and imaging with emission in the NIR region. [14,22,23] Furthermore,t he structural diversity of SWCNTs promises tunable emission wavelengths. [10,12] SWCNTs are highly sensitive to environmental changes, which is the basis for molecular recognition and was pioneered by optical sensors for glucose detection and DNAp olymorphism. [24,25] Both covalent or noncovalent functionalization approaches play an essential role in tailoring molecular interactions close to the SWCNT surface. [10,14,17,23,26] By using such concepts,S WCNT-based bio-sensors for many highly important biomolecules have been developed.
More recently,t his allowed chemical signaling to be mapped in ac ompletely new manner,f or example,r elease patterns of neurotransmitters from cells with high spatial and temporal resolution, which provides unique insights into fundamental biological questions. [27,28] Moreover,r ecent advances have been made in remote in vivo biosensing applications by the multimodal optical detection of several analytes.B yc ombining multiple nanosensor elements and integrating them into functional arrays,a nalytes can be identified and distinguished on the basis of their characteristic image signatures. [29] Such ac ombination of optical nanosensors could pave the way for the next generation of fast and reliable in situ diagnostics.I na ddition, these approaches provide completely new opportunities for standoff process controlling,f or example,f abrication of antibodies or monitoring in food and agriculture industries (smart plant sensors). [3,19,[30][31][32][33] In this Review we focus on optical biosensing with SWCNTs to give an update on this fast-evolving field. We evaluate in detail the specificity,sensitivity,spatial resolution, and biocompatibility of different SWCNT-based biosensors. This Review follows on from previous reviews, [2,10,14,23,34,35] Biosensors are powerful tools for modern basic researchand biomedical diagnostics.Their development requires substantial input from the chemical sciences.Sensors or probes with an optical readout, such as fluorescence,offer rapid, minimally invasive sensing of analytes with high spatial and temporal resolution. The near-infrared (NIR) region is beneficial because of the reduced background and scattering of biological samples (tissue transparency window) in this range.I nt his context, singlewalled carbon nanotubes (SWCNTs) have emerged as versatile NIR fluorescent building blocks for biosensors.H ere,w eprovide an overview of advances in SWCNT-based NIR fluorescent molecular sensors.W efocus on chemical design strategies for diverse analytes and summarize insights into the photophysics and molecular recognition. Furthermore,different application areas are discussed-from chemical imaging of cellular systems and diagnostics to in vivo applications and perspectives for the future. and discusses new chemical strategies developed in the last few years.S WCNTs can also serve as NIR labels.H owever, this is not discussed here and we refer to other excellent reviews. [1,2] In Section 2, the basic structural properties and photophysics of SWCNTs as well as functionalization strategies are described. To conclude this section, we touch on the most important aspects of SWCNT biocompatibility.S ection 3 contains an overview of general chemical recognition strategies.W ep rovide ad etailed and up-to-date summary of all currently accessible biomolecular target groups,i ncluding reactive oxygen species (ROS), neurotransmitters,p roteins, antibodies,lipids,and sugars.T his overview is complemented with mechanistic insights into how these sensors work. Finally, we provide ap erspective on the field (Sections 3a nd 4) and discuss possible future directions.T his includes novel biological topics such as plants,a dvanced chemical tools (defects), methods for improved (hyperspectral) imaging,novel screening approaches,a nd multiplexing.

Functionalization Concepts
Since the report of their structure,C NTs have attracted wide interest within the scientific community and beyond. Their remarkable mechanical, electrical, and photophysical properties have paved the way for applications in the fields of advanced materials,m icroelectronics,b iosensing,i maging, drug delivery,a nd many more. [8,36] Here,w ew ill briefly describe the structure and photophysics of SWCNTs,followed by approaches to tailor their surface chemistry and biocompatibility.

SWCNT Structure and Photophysics
CNTs can be conceptualized as rolled-up cylinders of graphene. [37] Their properties are determined by the exact sp 2hybridized carbon lattice as well as by the number of cylinders that are stacked into each other. [37] CNTs derived from as ingle graphene cylinder are called single-walled carbon nanotubes (SWCNTs), [38] whereas tubes consisting of multiple layers are called multiwalled carbon nanotubes (MWCNTs). [7] SWCNTs are commonly labeled using the chiral index (n,m), where n and m are integers that describe the carbon lattice structure (Figure 1a). [37,39] In this notation, the SWCNT is conceptually rolled up along the vector c = na 1 + ma 2 (a 1 and a 2 are the graphene lattice vectors). Consequently,t he roll-up vector also determines the diameter. ForSWCNTs,the reported diameters range from 0.4 nm to 10 nm. [8,40] Ther oll-up vector affects the density and energy of the electronic states of SWCNTs and consequently the optoelectronic properties are directly related to chirality.A sar esult, for nÀm = 0(armchair configuration), SWCNTs are metallic, for nÀm = 3j (j 2 Nn 0 fg ), semimetallic,a nd semiconducting for all other (n,m)c hiralities. [10] When SWCNTs are excited with light, an electron-hole pair (exciton) can be created and diffuses along the SWCNT axis. [41] Fors emiconducting SWCNTs, [18] the absorption of photons with energies corre-Julia Ackermann received her M.Sc. in nanoengineering with afocus on nano-(opto)electronics at the universityo fDuisburg-Essen (Germany). Since 2020 she has been persuing her Ph.D. in the technology division of biomedicaln anosensors of the Fraunhofer Institute for MicroelectronicC ircuits and Systems in Duisburg (Germany). Her work focuses on improving the overall selectivity of carbon nanotube based sensors by pattern recognition.
Justus Metternich received his Bachelor's degree in biotechnology from the University of Applied Sciences Darmstadt. After ashort stay at the Centro de Investigaciones Biológicas Margarita Salas in 2018, he continued with his Master's studies in chemistry at Uppsala University.S ince November 2020, he has been part of the Attract group of Sebastian Kruss and pursuing his Ph.D. at Ruhr-University Bochum. His research focuses on the design of fluorescentc arbon nanotube functionalizations for the detection of pathogens.
Svenja Herbertz received her Ph.D. in Physics at the Solid-State Physics Laboratory at the Heinrich-Heine-University in Düsseldorf (Germany) in 2019. During her years of study in Medical Physics she gained experience in the field of optical spectroscopya nd use of semiconductor quantum dots for fluorescencel abeling in biomedical applications. She is currently aresearcher at the Fraunhofer Institute for MicroelectronicC ircuits and Systems in Duisburg (Germany)i n the technology division of biomedicaln anosensors.
Sebastian Kruss received his Ph.D. in biophysical chemistry at Heidelberg University and the Max Planck Institute for Intelligent Systems (with Prof. Joachim Spatz). He then moved to the Massachusetts Institute of Technology (with Prof. Michael Strano), where he worked on carbon nanomaterials. After heading an independent research group at Gçttingen University (2015-2020), he became professor of physical chemistry at Ruhr-UniversitätB ochum and Attract group leader at Fraunhofer IMS. His research focuses on novel materials, fluorescencespectroscopy and microscopy,biosensors, and cell biophysics.
sponding to the visible spectrum of light typically leads to excitation to the second conducting band (Figure 1b). [9,12] Fast decay (femtosecond time scale) to the first conduction band followed by radiative recombination, then causes fluorescent emission in the NIR region (> 870 nm), [18,42] aregion that is particularly interesting for biological imaging (Figure 1c). Quantum chemical considerations predict 4 singlet and 12 triplet excitonic states. [43,44] However,o nly the transition from the singlet state is optically allowed. [43,44] As the energy of this state is higher than the majority of other singlet and triplet states, [43,44] av ariety of dark exciton decay pathways exist. [45] For(6,5)-SWCNTs,the size of an exciton is approximately 2nm. [46] During their lifetimes they diffuse in the range of 100 nm along the SWCNT axis. [47,48] As all carbon atoms are located on the surface of the SWCNT,excitons are affected by the nanotube corona (i.e.t he organic phase around the SWCNTs). Consequently,t he photophysics of SWCNTs are highly influenced by chemical processes around their surfaces.T his renders them ideal building blocks and transducers for chemical and biological sensing.

Surface Functionalization
Theextended p-system makes SWCNTs hydrophobic and consequently they easily aggregate in solvents like water. Therefore,a ni mportant step in the preparation of SWCNTbased sensors is their functionalization to isolate,s olubilize and colloidally stabilize single SWCNTs.T he functionalization also serves the purpose to a) interact (specifically) with other molecules and b) translate this interaction into af luorescence change.
In the past years,d ifferent covalent and noncovalent modification strategies ( Figure 2) have been developed. For acomplete overview,werefer the reader to several excellent reviews and discuss only concepts relevant for sensing here. [26,[51][52][53] On am ore abstract level, two strategies to assemble selective SWCNT-based sensors have been used, namely screening and rational design (Figures 3and 5). Thefirst one relies on permutations of the organic corona around the SWCNT (e.g.d eoxyribonucleic acid (DNA) sequence) whereas the second one uses known recognition motifs (e.g. antibodies).

Noncovalent Functionalization
Noncovalent functionalization in aqueous solution is achieved by sonication with surfactants that form micellar structures around the SWCNT or through strong p-p interactions with the SWCNT surface ( Figure 2). Prominent examples of surfactants are sodium dodecylsulfonate (SDS), sodium dodecylbenzenesulfonate (SDBS), sodium cholate (SC), sodium deoxycholate (DOC), lithium dodecyl sulfate, Tr iton X-100, and pluronic F127. [17,26] Additionally,functional surfactants-for example,with aperylene core together with ah ydrophilic dendron-adsorb through p-p stacking and enable energy transfer. [54] In general, as urfactant concentration above the critical micelle concentration is required to stabilize dispersed SWCNTs in solution. [17] Thus,t hese approaches are limited with regards to experiments in complex (biological) systems.
In contrast, functionalization with larger biopolymers enables the formation of stable conjugates.H ere,D NA and ribonucleic acid (RNA) form strong p-stacking interactions between the nucleobases and the SWCNT surface,t hereby exposing their negatively charged phosphate backbones and solvating the SWCNT-nucleic acid complex ( Figure 2). [22,55] As the conformation of the SWCNT-nucleic acid complex is affected by changes in the local ion concentration, [25,56,57] locked nucleic acids have been used as more rigid synthetic derivatives at higher salt concentrations. [57] As alternative to nucleic acids, [25] certain polycyclic aromatic compounds carrying hydrophilic moieties have effectively solubilized SWCNTs.Similar to the p-stacking [51,58] of those compounds,t he functionalization of SWCNTs with peptides, [59,60] proteins, [60,61] and other polymers [62,63] has been widely demonstrated ( Figure 2). SWCNT-based biosensors have been rationally designed by the attachment of antibodies (or analogues;F igure 4a,b,d) [64,65] and peptides (Figure 4e) [66,67] to polymers or by the adsorption of boronic acids (Figure 4c) [68] and aptamers (Figure 4f)onSWCNTs. [27] In cases when sonication would destroy the structural integrity of the (bio-)polymers,p rimary suspension of the SWCNTs in asurfactant, followed by subsequent exchange to the polymer by dialysis has been employed. [24,60,69] An alternative to this rational design is the screening/search for b) The band gap structure gives rise to fluorescence emission in the near-infrared (NIR) region. c) The E 11 transition of SWCNTs [49] overlaps with the tissue transparency window,thus offering the advantage of reduced light absorption, [50] scattering(e.g. Rayleigh), and background fluorescence. Here, the emission spectrum of SWCNTsof( 6,5)chirality is shown, but the emission wavelength for other chiralities span the whole NIR region. novel organic phases.T his concept was named corona-phase molecular recognition (CoPhMoRe). [70] Here,aheteropolymer adsorbs onto the carbon nanotube surface and forms an ew structure (corona) that serves as am olecular recognition site for interaction with an analyte.T he biomolecules used are typically amphiphilic with hydrophobic domains that enable SWCNT adsorption and hydrophilic domains to be responsible for the entropic stabilization of the SWCNT in suspension and formation of abinding site for the analyte. [70] It is important to note that the biomolecules/polymers alone do not necessarily need to interact selectively with the analyte of interest. [34,70] As such, the formation of these recognition sites cannot be predicted and are typically found by screening or high-throughput approaches.P rominent examples of CoPhMoRe screenings are the identification of SWCNTbased neurotransmitter sensors [15] as well as the adaptation of the CoPhMoRe concept to proteins [71] (Figure 5).

Covalent Functionalization
Thec ovalent functionalization of SWCNTs introduces new s-bonds into the sp 2 -hybridized SWCNT structure.I n contrast to noncovalent functionalization methods,t he conjugates promise higher stability. [10] However,the uncontrolled introduction of covalent sp 3 bonds (defects) destroys the electronic and optical properties and diminishes the intrinsic NIR fluorescence. [10] One strategy to overcome this problem preserves the sp 2 -hybridized structure of SWCNTs during their covalent functionalization. [72] In contrast, ac ertain number of sp 3 defects give rise to novel properties,s uch as red-shifted emission features that are capable of single photon emission. [53,[73][74][75] Therefore,t hese defects are also called quantum defects or quantum color centers. [53] These sp 3 defects have,a tl ow densities,b een shown to increase the fluorescence of SWCNTs. [53,74,76] Incorporation of these defects at low concentrations leads to the trapping of excitons and an alternative decay pathway (E 11 * )t hat results in an ew red-shifted fluorescence feature (Figure 6a). [74,76] A wide range of sp 3 defects has been incorporated into SWCNTs to increase the fluorescent properties by using diazo ether, aryl halide,(bis-)diazonium, as well as Billup-Birch and alkyl halide reductions. [53] Additionally,O-doping approaches using . Rational molecular recognitionc oncepts in SWCNT-based biosensors. a) Conjugationo fHis-tagged troponin antibodies to SWCNTs wrapped with Ni 2+ -chelating chitosan for the detection of the cardiac biomarker troponin. Adapted from Ref. [64] with permission. Copyright 2014 John Wiley and Sons. b) Attachmento fananobody against the green fluorescentprotein (GFP) to DNA-functionalized SWCNTstotarget GFPtagged proteins in vivo. Adapted from Ref. [47] with permission.C opyright2 019 John Wiley and Sons. c) Adsorbed aryl boronic acids react with sugars, which modulates the SWCNT fluorescence. Adapted from Ref. [68] with permission.C opyright 2012 AmericanC hemical Society. d) Antibody-DNA-SWCNT complex for the detectiono fthe ovarian cancer biomarker human epididymis protein 4(HE4). Adapted from Ref. [65] with permission.e)Short peptides conjugated to DNA adsorbedo nSWCNTsenable the binding of cell adhesion receptors. Adapted from Ref. [66] with permission.C opyright 2018 American Chemical Society.f)Serotonin-binding aptamersonSWCNTsenable the detection of serotonin release from cells. Adapted from Ref. [27] with permission.C opyright 2019 AmericanC hemical Society. Figure 5. Screening approaches based on corona phase molecularr ecognition (CoPhMoRe).a )The screening of SWCNT-polymer conjugates (x axis, N1-N13:n ucleic acids;PL1-PL12:p hospholipids;P 1-P5:amphiphilicp olymers) identifies SWCNT-basedsensor candidates with astrong fluorescence in response to different neurotransmitters (y axis). Reprinted from Ref. [15] with permission.C opyright 2014 American Chemical Society.b )CoPhMoRe screening procedure of SWCNT-polymer conjugates (x axis) for the detection of proteins (y axis). Reprinted from Ref. [71] with permission.
ozone and light, [75] sodium hypochlorite, [77] as well as hydroperoxides of polyunsaturated fatty acids [78] have been reported to increase the red-shifted emission of SWCNTs at low defect concentrations ( Figure 2). Covalent functionalization approaches also offer opportunities beyond changes in the photophysics.Defects that can be further functionalized enable the conjugation of important biomolecules.R ecently,m aleimide defects were used to link proteins such as nanobodies and phenylalanine defects to grow peptides directly on the SWCNT surface,s imilar to as olid-phase peptide synthesis [79] (Figure 6b). Thec ovalent conjugation approach with dichlorotriazine allows subsequent nucleophilic aromatic substitution of the chlorides using amine-containing linkers [72,80] (Figure 2). Another example are defects that are already able to interact with other biomolecules,such as phenyl boronic acids that interact with saccharides,a nd change the E 11 * (S 11 * )a nd E 11 (S 11 ) emissions. [81] It is interesting to note that the resulting bathochromic shifts (E 11 * )c aused by sp 3 defects can be tuned using the electronic properties of the incorporated moieties. [74,76] That said, defects provide ar ich chemical playground and interested readers are referred to several excellent reviews. [53,82] With regards to SWCNTs with aryl defects,e lectron-withdrawing substituents generally introduce red-shifts to the E 11 * emission that can be correlated to the Hammet constants (s) of the substituents. [74,83] Furthermore,the E 11 * red-shift shows a1/d 2 dependence on the diameter (d)ofthe SWCNT. [74] The protonation of diethylamino-substituted aryl defects (s HR2N+ =+0.82 vs. s R2N = À0.66) [84] is ag ood example of the effect caused by the inductive effects of substituents. Furthermore,t his type of defects allows ap recise sensing of Figure 6. Covalent functionalization of SWCNTs. a) The controlled introductiono fsp 3 defects creates an alternative decay pathway that brightens dark excitons without destroying the normal E 11 NIR fluorescence. Reprinted from Ref. [74] with permission.C opyright 2013 Nature Publishing Group. b) Introduction of certain aryl defects as generic handles to functionalize SWCNTsw ith biomolecules. Adapted from Ref. [79] with permission.c)The protonation of covalently attached aminobenzenegroups modifies the energy level of the sp 3 defect state and changes the photoluminescence. Adapted from Ref. [85] with permission.C opyright 2015 American Chemical Society.d)Quantum defects change the fluorescence response of DNA-functionalized SWCNTstothe important biomolecule and neurotransmitter dopamine. (GT) 10 -functionalized pristine SWCNTs(pSWCNT) increase their fluorescence in response to dopamine. The same SWCNTswith sp 3 defects (dSWCNT) decrease their fluorescence, which shows the strong impact of defects on the sensing mechanism. Adapted from Ref. [87] with permission.C opyright 2021 American Chemical Society. the pH value down to 0.2 units through the changes in the E 11 * emission ( Figure 6c). [85] Apart from the introduction of defects for sensing,t he covalent functionalization of SWCNTs can be used for site assembly using different (bio-)polymers and linkers. [86] Defects also change the exciton decay pathways and, therefore,a ffect the photophysics and sensing mechanism of SWCNT-based sensors.T his approach was recently used to perturb the sensing and elucidate the rate constants that are involved [87] (Figure 6d).
In this study it was also found that as mall number of defects can inverse the sensing response from as trong increase to as trong decrease in fluorescence.

SWCNT Biocompatibility
Biocompatibility is highly important for materials in direct contact to biological matter.E ven though many biocompatibility studies exist, the conclusions are difficult to compare. [88] Them ain reason is that different materials, surface reactions,and biological systems are compared, which leads to anoncoherent view.Moreover,the SWCNT field has evolved dramatically over the years and well-defined chirality pure SWCNTs-based sensors with ultrahigh purity are available today,w hereas older studies used less well-defined materials. [49] In addition, the application itself determines the perspective. [89] As aresearch tool, SWCNTs should not affect the biological system in such away that the results are biased. Forlong-term in vivo studies and applications in humans,the fate of SWCNTs in biological systems is highly relevant for their biocompatibility.S WCNTs have been shown to be susceptible to degradation by oxidative processes introduced by neutrophils [90] and macrophages. [91] Furthermore,t he functionalization changes the surface properties of the nanotubes and, thus,ultimately the way SWCNTs interact with the molecules in a( biological) system. [92] Fore xample,e ndocytosise xperiments have shown that the DNAs equence length plays an important role in endocytosis and retention time scales of DNA-functionalized SWCNTs within mammalian cells. [93] Additionally,e xperiments using acombination of NIR fluorescence spectroscopy and resonance Raman scattering have been used to analyze the fate of DNA-functionalized SWCNTs through the endosomal process. [94] Based on the experimental findings,t he authors propose that DNA-SWCNTs enter the cell, where they are transported into early endosomes.Maturation of the endosome begins with ad ecrease in the luminal pH value, which is followed by as eries of physicochemical processes that transform the endosome into al ysosome,w here the SWCNTs finally aggregate. [94] As correct functionalization has been shown to alleviate the pathogenicity of SWCNTs, [95] stable functionalization is one possible way to safeguard the future design of SWCNTbased sensors in environments where long-term stability is of the highest importance.Adequately functionalized SWCNTs have been shown to possess excellent biocompatible properties.Agood example is the recently published long-term biodistribution and compatibility assessment of DNA-encap-sulated SWCNTs after intravenous administration in mice. [96] After an initial increase in the SWCNT fluorescence in the liver,t he SWCNT fluorescence decreased rapidly over the course of 14 days. [96] Thes ame trend is also seen in the longterm SWCNT biodistribution in different organs.B yu sing hyperspectral microscopy,l ow levels of SWCNTs were detected in murine hearts,l ungs,l ivers,k idney,a nd spleen tissues one month after injection. Assessment of these tissues after three and five months showed no SWCNT fluorescence in lung tissue,o ri nh eart and lung tissues. [96] Moreover,n o abnormalities were found in chronically exposed tissues after hematoxylin and eosin (H&E) staining at all observed time points and the assessed biomarkers showed negligible changes up to four months,a nd minor changes after five months. [96] Thea forementioned studies suggest remarkable opportunities for SWCNTs in biomedical applications.A sac onsequence of the interplay between different materials,surface reactions,a nd biological systems it becomes evident that biofunctionalized SWCNTs represent ac lass of different materials.A sf or all new materials,t he biocompatibility should be evaluated for every type of chirality,p urity, functionalization, and route of administration. [96] Thes cientific community is well-aware of this problem and it has been pointed out that an assessment of these parameters in the context of biocompatibility depends on the context of the experiment, timescale,a nd the application of the nanomaterial. [88,97] As ac onsequence,i ti sf undamentally important to place experimental data in the right context. [88] An important requirement for ab iocompatible design of SWCNT-based sensors is an in-depth understanding of the composition of the protein corona in biological media. In this regard, arecent study characterized the enrichment of certain proteins in the SWCNT corona. [98] In the future,l ong-term studies comparing the biocompatibility of different SWCNT subclasses (purity,c hirality, surface chemistry) would be desirable to safeguard the development of biocompatible sensors.Afoundation for the standardization of protocols could be the MIRIBEL (Minimum Information Reporting in Bio-Nano Experimental Literature) reporting standard. [99] As the functionalization of the SWCNT plays acritical role in the biocompatibility of SWCNT-based sensors,t he design of stable SWCNT functionalizations needs to be carefully ensured for long-term applications.I np articular, the recent advances in covalent functionalization strategies might, therefore,offer interesting starting points for future development. [79] 3. SWCNT-Based Sensors

Development of Chemical Design Strategies
Thed iscovery of band gap fluorescence from SWCNTs and their structure-dependent NIR emission wavelength marks the starting point for SWCNT-based sensors. [12,18] Given the high surface to volume ratio of SWCNTs,i tw as quickly anticipated that SWCNT fluorescence would be sensitive to the chemical environment. [24] Thefirst generation of sensors targeted mainly smaller molecules including protons and reactive oxygen/nitrogen species (ROS/RNS). In these cases,t he fluorescence changes were most likely caused by direct quenching. At the same time,i tw as discovered that different surface reactions with biopolymers lead to molecular interactions that are surprisingly specific even without using astandard approach with antibodies.This idea was conceptualized as corona-phase molecular recognition (CoPhMoRe). [70] During the last few years,g reat progress has been made in the chemical design of sensors for both biomedical and environmental applications.I nt he next sections we will give an overview on different sensing strategies,o rganized according to the molecular target. Here,sensing of the target molecule has partly environmental as well as biomedical applications.W efocus on the advances in the last few years but also report previous studies (see  in the Appendix).

Biosensing of Target Analytes
Theongoing advances in the development of recognition strategies have led to powerful biosensors based on SWCNTs. Va rious targets can be detected with high selectivity and sensitivity by combining arecognition unit with the SWCNTs. Recognition strategies ( Figure 3) can mainly be categorized into ascreening ( Figure 5) and arational approach (Figure 4). Thef irst approach is in principle achieved with al ibrary of synthetic organic phases (coronas) consisting of different amphiphilic polymers wrapped around SWCNTs and screened against apanel of various analytes to find aselective interaction. [15,70,71,100,101] Thelatter approach is mostly applied for the detection of larger molecules such as proteins [65,[102][103][104][105][106] or sugars [11,107,108] by conjugating ak nown binding partner of the target analyte to the SWCNT surface.Several approaches are based on the use of SWCNTs wrapped by single-stranded DNA( ssDNA), whereby different lengths of the (GT) x sequence is probably the most used sequence up to now.

ROS/RNS
ROS/RNS are important signaling molecules in many organisms, [122] but their detection is challenging because they diffuse fast and have short lifetimes due to their high reactivity with O 2 and other molecules. [10,123] Since the finding of the first NO sensor based on SWCNTs [124] the performance of SWCNT-based ROS sensors has grown from the first selective detection of NO and H 2 O 2 at the single molecule level [13,125,126] and first in vivo applications [127] to an ew approach to study NO generation and spatiotemporal imag-ing of intracellular NO signaling. [128] Recently,amathematical model that calculated the NO concentration based on the change in the SWCNT fluorescence was derived. [129] This was previously not possible due to an onlinear fluorescence quenching rate in response to NO. [13] ROSp lay am ediating role in the cell-to-cell communication of plants to activate defence mechanisms, [122] whereby it has become clear that H 2 O 2 is the primary mediator that responds to different stresses in plants. [130] This has led to novel SWCNT sensor approaches to study ROS within plants. [19,20,31,32] Wu et al. demonstrated remote H 2 O 2 monitoring of plant health with sensitivity in the plant physiological range by using fluorescent SWCNT-based sensors. [31] Their rational approach was based on aD NA aptamer that specifically binds to the porphyrin hemin (HeAptDNA-SWCNT). Hemin binds ferric ions,w hich undergo aF entonlike reaction with H 2 O 2 to produce hydroxyl radicals (Figure 7a)t hat directly quench the SWCNT fluorescence.F or spatiotemporal in vivo monitoring,SWCNTs were embedded in leaves of plants and the plants exposed to different stresses such as UV-B,h igh light intensities,a nd ap athogen-associated peptide (flg22; Figure 7b). Thedecrease in fluorescence reported remotely different aspects of the stress.T hese differences in fluorescence intensity quenching offer the possibility to interpret stress patterns in plants.
Similar to this approach, Lew et al. developed aplatform for H 2 O 2 detection in leaves of different plant species. [32] This sensor platform used ar atiometric approach, with (GT) 15  In the same manner,L ew et al. developed aS WCNTbased sensor system for the specific detection of arsenite in plants to monitor the uptake of the toxic heavy-metal pollutant arsenic by using as elf-powered microfluidic system in real time. [33] Fort his purpose,t hey infiltrated the sensors and the invariant reference into leaves of spinach, rice plants,a nd hyperaccumulating fern, which is able to preconcentrate and extract arsenic from soil ( Figure 8a). The intensity of the sensors increased steadily over several days, with the sensor response of the hyperaccumulating plant being significantly higher than those of the rice and spinach plants (Figure 8b). Based on ak inetic model, the arsenite concentration in the leaf and the limit of detection (LOD) were calculated to be 4.7 nm and 1.6 nm as af unction of the root fresh weight and uptake solution volume after 7a nd 14 days (Figure 8c). These examples show that SWCNTbased H 2 O 2 sensors are able to report plant stress on am icroscopic and macroscopic level with potential applications in smart agriculture.
Another macroscopic situation in which H 2 O 2 plays an important role is wound healing. Safaee et al. developed aw earable optical microfibrous material with encapsulated SWCNT-based sensors ( Figure 8d)t om onitor the H 2 O 2 concentration in wounds. [112] Their approach was based on the ratiometric signal of (8,7)/(9,4)-SWCNT chiralities,which differed in their response to H 2 O 2 ( Figure 8e). Thef luorescence signal was invariant to the excitation source distance, and exposure time,w hich enabled detection within commercial wound bandages with aw ireless readout (Figure 8f). These microfibers encapsulated the SWCNTs for at least 21 days without structural changes.
Furthermore,Z heng et al. reported the selective interactions of SWCNTs coated with ten different ssDNA sequences in response to dissolved oxygen. [131] TheS WCNT emission intensity was quenched between 9t o4 0% depending on both the ssDNAs equence and SWCNT chirality in response to 1atm O 2 compared to samples purged with 1atm argon, thus indicating that stronger coating interactions lead to reduced O 2 access to the SWCNT surface.S ince the quenching reversed completely after the removal of dissolved oxygen, it is probably based on physisorption on the SWCNT. Thus,t he screening for fluorescence quenching by dissolved oxygen provides as imple approach to explore the structureselective interactions of ssDNAwith SWCNTs.
ROSc an also be generated by enzymes and SWCNTs. Yaari et al. demonstrated the first SWCNT-based sensor that reports the degree of enzymatic suicide inactivation. [201] The approach was based on enzyme-bound SWCNTs,w hich report fluorescence modulations by quenching and redshifting selectively in response to substrate-mediated suicide inactivation of tyrosinase.M echanistic insights revealed that the red-shifted response is most likely ar esult of the generation of singlet oxygen during the enzymatic reaction, which leads to the binding of ssDNAo nt he SWCNT surface. [132]

Neurotransmitters
Neurotransmitters are an important class of signaling molecules.T ou nderstand neuronal networks and linked neurological diseases,i maging with high selectivity and spatiotemporal resolution is necessary,w hich existing methods are currently not able to provide. [133] In the last few years Figure 7. In vivo monitoring of plant health. a) SWCNTsfunctionalizedw ith aDNA aptamer for hemin (HeAptDNA-SWCNT) serve as asensor for H 2 O 2 ,which is an important signalingm olecule for plant stress. Hemin catalyzes the reaction of H 2 O 2 to hydroxyl radicals, which quench the fluorescence of the SWCNT.Spatial and temporal changes in the NIR fluorescence intensity in leaves embedded with HeAptDNA-SWCNT sensors are remotelyr ecorded by aN IR camera to assess plant health. b) The sensor's NIR fluorescence decreases reversibly in the presence of the peptide flg22, which mimics apathogen attack. Reprinted from Ref. [31].C opyright 2020 AmericanC hemical Society.c )SWCNTsf unctionalized with (GT) 15 -ssDNA (G-SWNT) respond to H 2 O 2 .d)Bright-fieldi mage of aspinach leaf infiltrated with G-SWNT (left) and nonresponsive A-SWNT (right) in combination with af alse color plot after wounding shows that only the G-SWNT spot decreases in intensity.e )Ratiometric sensor response after application of different types of stress. Reprinted from Ref. [32] with permission.C opyright 2020 Springer Nature.
several SWCNT-based sensors based on functionalization with DNAh ave been explored and it has been shown that sensitivity and selectivity depend on the exact DNA sequence. [100,118,119] Thef irst SWCNT-based sensors for the detection of neurotransmitters were reported by Kruss et al. [15] By using as creening approach, it was found that certain ssDNA-SWCNTs change their fluorescence in the presence of catecholamine neurotransmitters such as dopamine.T hese sensors were reversible and showed sensitivities in the nanomolar range.S imilar sensors were used for the highresolution imaging of cellular dopamine efflux from stimulated neuroprogenitor cells. [100] Here,t he sensors were immobilized on ac ollagen-coated glass substrate to increase cell adhesion, and dopamine-releasing neuroprogenitor (PC12) cells were cultured on top.Inresponse to astimulation event, the fluorescence intensity of the sensor layer image consisting of up to 20 000 pixels increased (Figure 9a,b). This allowed both the spatial and temporal dynamics of dopamine release events to be studied with extraordinary high resolution and to identify hotspots (Figure 9c,d).
This approach of imaging many nanosensors under cells is also applicable to other neurotransmitters.D inarvand et al. imaged the release of serotonin from human blood platelets in real time, [27] as most of the serotonin is stored in blood platelets and not in the brain of humans.This serotonin sensor (NIRSer) consisted of as erotonin-binding aptamer on aS WCNT,w hich exhibited an increased fluorescence emission of up to 80 %i nr esponse to serotonin (Figure 10 a). High-resolution images of serotonin release patterns from single cells were obtained by placing the sensors below and around serotonin-releasing cells (Figure 10 b,c). This approach allows serotonin release to be studied with unprecedented resolution and the time delay between stimulation and release to be resolved.
In as imilar fashion, artificially added serotonin was detected in acute brain slices by Jeong et al. [134] In this case, aD NA sequence was found by an expanded screening approach from alibrary of around 6.9 10 10 different ssDNA-SWCNTs.T hese ssDNA-SWCNTs exhibited as elective response to serotonin over serotonin analogues,m etabolites, and receptor-targeting drugs.
Theh igh spatiotemporal resolution of SWCNT-based neurotransmitter sensors is especially useful when it comes to resolving parallel processes on the subcellular to cell-network length scale.However, placing the sensors below cells can be adrawback, especially with cells that need to differentiate on this layer for several weeks.Elizarova et al. developed anew sensor paint approach (AndromeDA) to use sensors and study the dopaminergic signaling in primary neurons. [135] In this case,t he sensors were adsorbed ('painted')o nto the complex cell networks,w hich included different cell types. This approach allowed the heterogeneity of dopamine release events to be quantified from up to 100 release sites (varicosities), which is highly important to understand the information processing and plasticity of neurons.
An effect that has to be accounted for in these studies is that SWCNT fluorescence is affected by changes in the local cation concentration, which is also ah allmark of neuronal activity. [56,57] To circumvent this problem, Gillen et al. used   . High-resolution imaging of serotonin( 5HT) release from cells. a) NIRSer sensor:SWCNTsfunctionalized with aserotonin aptamer respond selectively to serotonin. b) NIRSer increases its fluorescence in response to serotonin. c) Sensors are immobilized on asurface and serotonin-releasing cells are cultured on top. Here, blood platelets are used, which contain most of the body's serotonin. d) Color-coded image of serotonin release from asingle platelet at three time points (before, during, and after serotonin release). e) Fluorescencer esponse from aregion of interest (ROI, green circle in (d)). The activation and delay time of the onset of serotonin release are marked with arrows. Reprinted from Ref. [27] with permission.C opyright2 020 American ChemicalS ociety. locked nucleic acids to develop sensors with improved stability to cation-induced fluctuations of the fluorescence intensity. [136] By systematically introducing locked bases along the (GT) 15 -DNAs equence they found that the fluorescence stability in the presence of Ca 2+ ions depends on the type of the locked bases.C ertain SWCNT chiralities exhibited improved stability against Ca 2+ ions and retained their ability to detect dopamine in the presence of Ca 2+ ions,t hus highlighting the importance of the exact conformation of the nucleic acid sequence.M oreover,t he detection of both Ca 2+ and dopamine was possible by monitoring multiple chiralities simultaneous.
Interestingly,t he fluorescence responses of SWCNTs suspended in sodium cholate to dopamine and serotonin can be altered by modulating the exposed area by the surfactant concentration. However,s uch surfactants would not be compatible with cellular systems. [137] Ac entral challenge in biomedicine is the controlled delivery (uptake,t ransport, and release) of (nano)materials such as sensors and pharmaceuticals.E ven whole cells can serve as vehicles to take up such materials.C ertain immune cells (neutrophils) have been shown to be suitable for cargo delivery by hijacking ap rocess known as neutrophil extracellular trap formation (NETosis). During NETosis,cells lyse after rupture of the cellular membrane by chromatin swelling. [138] Meyer et al. showed that human immune cells take up ssDNA-SWCNT-based sensors and can be triggered to release the cargo after ac ertain time by using NETosis. [139] Moreover,t he sensors maintained their functionality to detect dopamine and H 2 O 2 ,w hich offers opportunities for in vivo delivery.
All these discussed neurotransmitter sensors responded by af luorescence increase.I nterestingly,t he introduction of as mall number of aryl defects into ssDNA-functionalized SWCNTs completely reversed the sensing response (Figure 6d). [87] TheE 11 emission slightly decreased and strongly decreased the red-shifted E 11 * emission. Apart from new insights into the sensing,t his approach enables ratiometric detection schemes.F or am ore detailed overview on the biological relevance of catecholamine neurotransmitters and alternative detection methods (e.g.e lectrochemical) readers are referred to the literature. [28,114]

Other Small Molecules
Beyond neurotransmitters,r ecognition strategies for other small molecules have been developed, for example, adenosine 5'-triphosphate, [140] nitroaromatics, [30,141] riboflavin, [49,70,142] l-thyroxine, [70] oestradiol, [70] doxorubicin, [115,143] and steroids. [144] Finding as pecific molecular recognition element is difficult for many of these biomolecules,s uch as hormones, due to their chemical similarity.T herefore,L ee et al. used apolymer self-templating synthetic approach, which is based on the attachment of ac hemical appendage similar in molecular weight and structure to the target analyte to create ab inding pocket within the corona. This approach reduced the library size for screening and led to implantable SWCNT-based sensors for the selective detection of the human steroid hormones cortisol and progesterone. [144] Recently,t he first reversible fluorescent SWCNT-based sensor for volatile organic compounds was also reported, which has potential for the detection of wine spoilage. [145] For this,S humeiko et al. used peptide-encapsulated SWCNTs, which were adsorbed onto apolystyrene cuvette to detect low concentrations of acetic acid (down to 0.05 %( v/v)) in air. Using (6,5)-SWCNTs,w hich fluoresce below 1000 nm they demonstrated the detection with al ow-cost Si-based camera (Figure 11 a). Thes ensor was exposed to different concentrations of acetic acid, which quenched the fluorescence but was reversible when switching to clean air (Figure 11 b). The ability to identify wine spoilage was investigated by using two wine types with and without the addition of acetic acid to simulate an undesirably high acetic acid concentration (  In an extension of this study,this system was expanded to an array of five different peptide-encapsulated SWCNTs on an itrocellulose paper.T he optical patterns enabled the distinction of volatile molecules such as ethanol, methanol, and 2-propanol as well as the aromas of red wine,b eer,a nd vodka by linear discriminant analysis and machine learning. [146] One way to detect or study small molecules is to mimic parts of larger biomolecules,s uch as enzymes.D ong et al. screened alibrary of 24 amphiphilic polymers to find acorona phase which demonstrates abinding specificity very similar to the enzyme phosphodiesterase type 5( PDE5), which catalyzes the hydrolysis of secondary messengers. [147] The SWCNT-based sensor consisted of ap oly(methacrylic acidco-styrene) motif.This synthetic corona mimics the Hloop of the native enzyme and is,t hus,a ble to bind to Va rdenafil, aPDE5 inhibitor, and its molecular variant as aresult of the unique corona phase configuration. It is selective over other off-target inhibitors,b ut not completely over the chemically similar inhibitor Sildenafil.
One of the challenges in SWCNT-based sensing is the heterogeneous material that is used for most sensors.E ven though purification has made tremendous progress,g etting access to chirality-pure SWCNTs with at ailored surface chemistry has been ac hallenge.N ißler et al. showed sensing of small molecules such as riboflavin and ascorbic acid as well as pH value with chirality-pure SWCNTs by using aqueous two-phase extraction and asubsequent surface functionalization exchange process. [49] Thec hirality-pure sensors were up to ten-times brighter than mixtures of SWCNT chiralities,and enabled insights into the impact of chirality and handedness of SWNCTs and the sensing mechanism. Additionally,l ongtime stability over 14 days was demonstrated as well as ratiometric and multiplexed sensing based on the non-overlapping fluorescence spectra (Figure 22 b,c).
On am acroscopic level, monochiral SWCNTs were used by Nißler et al. to detect polyphenols in and around plants. [148] Polyphenols are secondary metabolites and messenger molecules that are released from leaves and roots as ac hemical defence against pathogens and herbivores (Figure 12 a). Certain polyethylene glycol phospholipids (PEG-PL) were identified for the selective detection of different polyphenols over interfering molecules such as sugars or H 2 O 2 .T he SWCNT-based sensors responded through quenching and red-shifting of up to 20 nm, for example,totannic acid (TaA, Figure 12 b). To image the plant polyphenol release over time, the sensors were embedded in agar,s oybean seedlings were plated on top,a nd polyphenol secretion was triggered with ap athogen-derived elicitor,w hich resulted in ad ecrease in the NIR fluorescence over time (Figure 12 c). These sensors help to understand this plant defence mechanism and could improve the breeding of stress-resistant plants for precision agriculture.

Lipids
Thei nvestigation of lipid-linked diseases is challenging because methods for accurate in vivo monitoring of lipid accumulation have been missing. TheH eller group was the first to address this issue by developing aS WCNT-based optical sensor, which non-invasively detects the lipid flux within the lumen of endolysosomal vesicles in vitro and in vivo. [117,149] In af irst approach, they used (GT) 6 -functionalized (8,6)-SWCNTs,w hich fluoresce at 1200 nm and responded through aw avelength shift to biological lipids and water-soluble lipid analogues. [117] By incubating the sensors with fibroblasts from al ysosomal storage disorder Niemann-Pick-type Cp atient in vitro, the sensors localized in the lumen of endolysosomal organelles without affecting their properties and resolved the lipid accumulation down to the subcellular level in real time.T he authors proved the reversibility of this sensor by administering adrug which reverses the disease phenotype.
Thes econd approach was based on the screening of several ssDNA-SWCNT chirality combinations to identify CTTC 3 TTC-(9,4)-SWCNTs with the greatest wavelength shift of up to 8nmi nr esponse to lipid accumulations. [149] The emission wavelength at 1125 nm is spectrally separated from the lipid absorption band at 1210 nm, thus facilitating promising in vivo applications.M olecular dynamics calculations led to the assumption that the lipid molecules sphingomyelin and cholesterol bind to the SWCNT surface by hydrophobic interactions,t hereby decreasing the water density in the SWCNT environment and leading to the observed blue shift (Figure 13 a). To validate the sensor functionality in live cells,e ndolysosomal lipid accumulation was induced in macrophage cells with chemical inhibitors to mimic different lipid phenotypes.A fter sensor incubation, ab lue-shift was observed for all sensors within the drugtreated cells compared to the control (Figure 13 b). Forinvivo applications,t wo mouse models were used with lipid accumulation within the organelles of many cell types,f or example,i nK upffer cells.B yi ntravenously injecting the SWCNT-based sensors,arapid decrease of the SWCNT fluorescence and consequently removal of SWCNTs from all parts of the mice except for the liver were observed. The sensors were also able to report uptake and endolysosomal lipid accumulation of oxidized low-density lipoprotein (oxLDL;F igure 13 c). Therefore,t hese types of sensors provide novel insights into the complexity of lipid metabolism and related health states.

Proteins
Proteins are one of the major biomacromolecules.C onsequently,t he study of protein-protein interactions helps to understand the function of proteins or to find novel drugs. [150] As discussed above,the detection of proteins with SWCNTs is either based on attaching ak nown natural recognition element [21,64,65,[102][103][104][105][106]151] or as ynthetic heteropolymer to the SWCNT surface ( Figure 3). [71,101,152,153] Such studies go beyond detecting the presence of aw hole protein.
Williams et al. developed the first label-free optical sensor for the detection of glutathione-S-transferase (GST) fusion proteins based on glutathione-(TAT) 6 -SWCNTs (GSH-DNA-SWCNTs;F igure 14 a). [154] Thes ystem used distinct differences in the emission wavelength and intensity of SWCNT chiralities in response to GST and, consequently,t he ratiometric intensity of two chiralities (8,6)/(9,1) (Figure 14 b). The functionality of this sensor in response to four different GST fusion proteins was tested, which all showed as imilar ratiometric response and aL OD in the low nanomolar regime.T his approach has potential for the tracking of protein expressions in real time.
Thed etection of insulin is essential for diabetes as it controls the glucose levels in blood. Thefirst optical SWCNTbased sensor approach for detecting insulin was realized in 2010 by Cha et al. [155] They used an insulin-binding aptamer, which showed ah ighly specific and sensitive decrease in the fluorescence intensity on forming ag uanine quadruplex. Furthermore,t hey incorporated these sensors in ac ollagen extracellular matrix and demonstrated sensor reversibility with enzymatic proteolysis and the detection of insulin secreted by pancreatic b-cells. [156] Recently,i nsulin detection was enabled by screening alibrary of PEG-conjugated lipids.Itwas found that the C 16 -PEG(2000 Da)-Ceramide-SWCNT complex causes as ignificant decrease in the fluorescence intensity in the presence of insulin compared to other relevant proteins present in human whole blood. [101] Additionally,n onspecific recognition mechanisms such as hydrophobicity or molecular weight were ruled out. Forexample,the sensor response to shorter insulin fragments was measured, however no correlation to the molecular weight was observed (Figure 14 c). Thea nalysis of insulin fragments was further supported by enthalpy measurements that showed no affinity of the PEG-conjugated lipid itself,w hich highlights that the exact CoPhMoRe phase bound to the SWCNT is responsible for the insulin binding.In more complex environments,s uch as serum, the sensor affinity was lower but still sensitive enough (Figure 14 d).
Thea pplication of sensors in complex biological samples is often challenging.T he spontaneous adsorption of proteins onto all kind of materials changes the actual corona structure and might affect sensing.T oi mprove the performance of sensors in those protein-rich environments,af undamental understanding of the interaction between sensors and their biological environment is necessary.P inals et al. addressed this issue by studying the protein corona formation on (GT) 15 -SWCNTs in cerebrospinal fluid and blood plasma by mass spectroscopy. [98] Their results showed strong binding to fibrinogen and other proteins involved in blood clotting, lipid transport, and complement activation. Thei dentification of interactions responsible for the formation of protein corona revealed that Figure 13. In vivo detection of lipid accumulation. a) Molecular dynamics simulationsofssCTTC 3 TTC-(9,4)-SWCNTst hat serve as asensor for the lipids cholesterola nd sphingomyelin through achange in the SWCNT emission wavelength. b) Bright-field images overlaid with hyperspectral NIR images of the sensors in RAW2 64.7 macrophageswith or without the chemical inhibitors U18666A,L alistat 3a2, and Imipramine that change the intracellularlipid levels. c) Hyperspectral NIR images of the sensor before and after injection of oxLDL in mice report the in vivo lipid status. Reprinted from Ref. [149] with permission.
the outer corona formation can be reduced by optimizing the electrostatic interactions through the sensor design and dynamic flow conditions (e.g.w ith lateral flow assays or microfluidic systems), while entropic calculations must be considered for the inner corona. This study highlights the urgent need to investigate sensors not only in simple buffers but biologically complex environments.M ost recently,E hrlich et al. developed ac omplementary approach using an insulin aptamer (found within the natural insulin gene promoter) functionalized to the SWCNT surface through a( AT) 15 ssDNAa nchor sequence. [158] In contrast to the synthetic PEGylated-lipid, which has no prior affinity to insulin, this aptamer possesses ak nown affinity to insulin. However,t he observed sensitivity was lower than the previous approach.
Most of the SWCNT-based sensors that have been discussed so far were measured in solution. Thei mmobilization of SWCNTs on different porous paper matrices,f or example,n itrocellulose,h as several advantages for ar obust assay. [116] Paper-based immobilization enabled analyte detection within non-aqueous solvents such as edible oil, which was previously not possible.T of urther extend this system the authors used wax to pattern hydrophobic regions onto the paper to create am ultiplexed one-dimensional sensor barcode consisting of different ssDNA-wrapped SWCNTs.
Another important class in biomedical diagnostics are antibodies.T he detection of immunoglobulin G( IgG) with SWCNTs was realized by using chitosan-wrapped SWCNT noncovalently modified with immunoglobulin-binding proteins. [104,151] Recently,K ozawa et al. designed af lexible fiber optic interface coupled to nanosensors that was capable of detecting the aggregation status of human IgG by reporting the relative fraction of monomers and dimer aggregates with sizes of 5.6 and 9.6 nm. [159] Fort his purpose,t he SWCNTbased sensors were incorporated into ah ydrogel (HG) and attached to the end of af iber waveguide.P roteins are also part of pathogens and consequently disease markers.P inals et al. developed aS WCNT-based sensor which is functionalized with the angiotensin-converting enzyme 2(ACE2), ahost protein which shows ah igh binding affinity for the SARS-CoV-2 spike protein (Figure 14 e). [157] At wofold NIR fluorescence increase was detected 90 min after the addition of the purified spike protein (Figure 14 f). Passivation with ah ydrophilic polymer was used to enable detection of the spike protein in saliva and viral transport medium.
However,a ntibodies are not always available for all targets.Inaddition, the development of new recognition units can be expensive and tedious,w hich is why new approaches are directed to the development of multiplexed sensor arrays [146,151] to overcome the limited selectivity of existing single sensors.R ecently,Y aari et al. developed aS WCNT solution-based sensor platform to detect multiple gynecologic cancer biomarkers in uterine lavage samples. [160] Thea rray consisted of eleven different ssDNA-SWCNT sensors,and the optical change in the intensity and wavelength was extracted for twelve chiralities present in the sample,which resulted in 132 individual ssDNA-SWCNT complexes.W ith machine learning algorithms aclassification accuracy (F1 score) of 0.95 was achieved. With retraining, this sensor platform may not be limited to the detection of cancer biomarkers.T he large variety of possible SWCNT chiralities in combination with unlimited SWCNT wrappings opens possibilities to meet the rising demand of new recognition strategies.

Sugars
Sugars are important building blocks and metabolites. Glucose,inparticular,isamajor target, for which continuous monitoring of the glucose level in blood is desired. SWCNTbased sensing ranges from the use of glucose-specific enzymes [24,161,162] or proteins [108] to the first affinity sensor based on the competitive binding between glucose and its polymer dextran. [11] Although improvements were made,the first approaches suffered from limited reversibility and/or physiological detection range.O ne sensor that meets the requirements is based on the functionalization of SWCNTs with glucose oxidase (GOX), ag lucose-specific enzyme. [161] Thea ddition of glucose causes an increase in fluorescence emission. Thep roposed mechanism is based on SWCNT fluorescence being quenched by defect sites on the SWCNT surface,w hich are hole-doped through oxygen adsorption. Thea ddition of glucose causes an oxidation of the GOX wrapping,which behaves as an electron donor and passivates the oxygenated sites of the SWCNT,t hereby resulting in afluorescence increase.T his effect is reversible by removing the glucose.The sensor showed responses to five other tested saccharides,but with the highest response to glucose.
Another recognition element for saccharides is phenylboronic acids,w hich have been used to functionalize SWCNTs noncovalently for the detection of sugars. [68] Recently,c ovalent aryl-boronic acid defects were also incorporated in SWCNTs. [81] Upon interaction with fructose and glucose,these sensors decreased in fluorescence intensity and the E 11 * signal shifted, which can be used for spectrally encoded sensing.

DNA/RNA
One of the most abundant and important types of biomacromolecules are nucleic acids that store and process genetic information. Using ac onstruct of ac omplementary capture sequence connected to a( GT) 15 -sequence serving as an anchor, thus providing colloidal stability,S WCNTs were recently used to detect hybridization events of microRNA and other oligonucleotides directly in serum, urine,a nd in mice in vivo. [163] Upon the addition of complementary nucleic acids,aspecific blue-shift for different chiralities upon hybridization was observed. Additionally,the sensor response was reversible through toehold-mediated strand displacement and the sensors possessed aL OD in the picomolar range.
Further development of this sensor led to the first SWCNT-based sensor for the detection of HIV in serum. [164] Harvey et al. discovered that SDS-denaturized serum proteins lead to an enhanced optical response of the SWCNTs in response to DNAhybridizations.T hey hypothesized that the addition of SDS ensured both the liberation of the viral RNA genome and the denaturation of the proteins which competitively bind to the freed surface of the sensor. Theinteraction leads to ab lue-shift in the SWCNT emission (Figure 15 a). This was first shown for hybridization with complementary target miR-19 DNAc ompared to control R23 DNA ( Figure 15 b). Ad ose-dependent enhancement of the blue-shift occurred in the region of the critical micelle concentration of SDS;thus,the denaturation of proteins by SDS is considered to involve an unfolding process of the tertiary structure of the protein to complete denaturation. As the SDS concentration was increased, the amount of denatured protein absorbed onto the SWCNT surface after hybridization of the DNA increased and saturated at 2% SDS.F or HIV detection, the recognition strategy was based on as ensor consisting of (GT) 15

Reviews
Acompletely different application of DNAchemistry on SWCNTs was established by Cha et al. Based on the consumption of chemical energy delivered by the RNA molecules,t hey developed as ynthetic motor that transports nanoparticles through the mechanical motion of DNAc onformation changes along the SWCNTs. [165] Movements of the motor of over 3 mmwith aspeed of 1nmmin À1 were observed.

Enzymes
So far, the detection of enzymes with SWCNTs has been shown for the characterization of proteases and DNases as well as cellulases and pectinases.T he hydrolytic enzyme activity was measured by Kallmyer et al.,who used hydrolytic enzyme wrapped SWCNTs that respond to the target enzyme with af luorescent intensity quenching because of the degradation of the enzyme-wrapping. [166] Different polymer wrappings consisting of polysaccharides and polypeptides were used to study cellulase,pectinase,and bacterial protease. Most recently,they used this approach to evaluate the enzyme activity in soil using al ow-cost multiplexed and portable fluorimeter able to perform the measurement outside the laboratory only minutes after extraction from the field. [167] As aconsequence of the fresh nature of the soil sample,field tests indicated activities an order of magnitude larger than those obtained in benchtop experiments.
Enzymes are also released by microorganisms,which can be used to fingerprint them. Nißler et al. chemically tailored SWCNTs to detect enzymes such as DNases and proteases. [29] Fortargeting extracellular proteases,SWCNTs were modified with bovine serum albumin (BSA), which serves as an enzymatic substrate,w hile SWCNTs were functionalized with calf thymus (CT) DNAf or reporting DNase Ia nd S. aureus nuclease activity.The sensors showed afluorescence decrease,most likely as aresult of decomposition of the BSA surface coating. These SWCNT-based sensors were further used for the discrimination of bacteria, which are known to alter their chemical environment through the release of signaling molecules,e nzymes,a nd metabolites (see section below).
In contrast, Shumeiko et al. used peptide-encapsulated SWCNTs,w hich also responded through af luorescence decrease upon enzymatic digestion of the SWCNT wrapping. [168] They utilized alow-cost paper-based dipstick system, with which they evaluated the trypsin activity in urine samples as am imic for acute pancreatitis,w here abnormal trypsin concentrations are common.
To study the enzyme myeloperoxidase,w hich is involved in the regulation of inflammation processes,H ee tal. used aratiometric system based on graphene oxide (GO) wrapped SC-SWCNT sensors and GO-wrapped carboxymethylcellulose (CMC)-SWCNTs as ar eference. [169] GO and SC-SWCNTs showed an opposed fluorescence signal in response to enzymatic degradation. Whereas the blue fluorescence intensity of GO was increased due to oxidation and degradation of GO leading to the formation of graphene quantum dots,t he NIR emission of SWCNTs decreased due to the generation of defects on the SWCNT surface.Incontrast, the CMC-SWCNT reference was almost stable as ar esult of the better surface protection of the CMC-wrapping.

Epitopes and Metabolites from Pathogens
Microbial infections are one of the major causes of mortality worldwide.C urrently,t he limited number of diagnostic methods in combination with increasing antibiotic resistances demonstrate the rising need for the rapid, contactless,a nd specific detection of pathogens such as bacteria. Theo ptical properties of SWCNTs promise advantages for pathogen detection. Bardhan et al. developed M13 bacteriophage functionalized SWCNTs (M13-SWNTs,F igure 16 a),w hich are able to distinguish F'-positive and F'negative bacterial strains by modulation of the fluorescence intensity. [170] TheM 13 bacteriophage has ak nown binding affinity to F'-positive E. coli strains.T herefore,t hey intramuscularly infected the right flank of living mice with E. coli strains,either F'-negative DH5-a strains (Figure 16 b, left) or targeted F'-positive JM109 strains (Figure 16 b, right) and observed a1 .6-fold intensity increase over the nonspecific DH5-a strains.Injection of PBS into the right flank served as acontrol. To extend this SWCNT-based label to awider range of bacterial strains lacking F'-pili, they additionally attached an antibacterial antibody on those M13-SWCNTs (anti-S. aureus-M13-SWNT) through as treptavidin-biotin reaction. This approach is aN IR labeling rather than sensing.I n contrast, Nißler et al. created ac oncept to remotely distinguish six important pathogens using an array of SWCNTbased sensors: [29] four for specific bacterial target detection based on ar ational approach and four generic lower sensitivity sensors together with an invariant reference consisting of NIR fluorescent Egyptian Blue nanosheets (Figure 17 a). [171] To overcome unspecific effects arising from the complex composition of bacterial media, the sensors were incorporated into ahydrogel (HG) array,inw hich the pore size was varied in accordance to the size of the analyte.F or the detection of small molecules (such as siderophores), HGs with al ow porosity were used, whereas HGs with ah igh porosity allowed large enzymes to diffuse to the sensors.T he sensors were exposed to clinical isolates of bacteria and the unique change in the fluorescence intensity for each sensor was monitored remotely.Within 24-72 h, aunique fingerprint in response to the tested pathogens was visible (Figure 17 b), which further allowed differentiation of the pathogens by principal component analysis (PCA; Figure 17 c). Besides this spatially encoding,spectral multiplexing was implemented to differentiate S. aureus and P. aeruginosa by using two monochiral SWCNT-based sensors with different wavelengths compared to the reference.M oreover,t he signal of the sensor could be detected through tissue to adepth of > 7mm, which highlights the potential for biomedical in vivo applications.

Mechanism of Fluorescence Modulation
In SWCNT-based sensors or probes,the SWCNTs serve as transducer elements that translate chemical changes caused by an analyte in the vicinity of the SWCNT into afluorescent signal. Thus,S WCNT-based sensing involves molecular recognition and signal transduction. Theprecise mechanisms are most likely different for different analytes and different surfaces.H owever,f rom the literature one can distinguish several possible generic mechanisms ( Figure 18).

Direct Quenching
Direct quenching,t hat is,t he decrease of fluorescence,i s caused by adsorption of the analyte onto the SWCNT surface. Changes in the pH value caused by the addition of an acid can cause protonation of the SWCNT sidewall and result in direct, reversible quenching of the SWCNT fluorescence (Figure 19 a). Mechanistically,t his can be explained by the injection of an electron hole into the p-system near the protonation site. [47] Excitons encountering such an electron hole will be quenched through an onradiative Auger process. [172] Furthermore,e lectron transfer between the valence band of 3,4-diaminophenyldextran-functionalized SWCNTs and the lowest unoccupied molecular orbital (LUMO) of nitrogen oxide results in rapid, reversible quenching of the SWCNT fluorescence. [124] Both of these interactions take part in the vicinity of the SWCNT and, consequently,s olvent effects should play alarge role.

Impact of Conformational Changes and Solvation
Within the dimensions of the exciton, the fluorescence of SWCNTs is very sensitive to the surrounding environment. To study the contributions of the solvent to the fluorescence of SWCNTs,f luorescence spectra were analyzed in different dielectric environments. [173,174] By using the solvatochromic shifts,asemiempirical scaling model was developed that linked optical with structural parameters and suggested an inverse dependence of exciton polarizability on the diameter and the square of the transition energy. [173] In nonpolar solvents,t he solvatochromic modulation of the fluorescence intensity becomes more pronounced for larger diameters. [174] Changes in the solvatochromic shift, on the other hand, were more pronounced for SWCNTs with smaller diameters. [174] In general, the displacement of H 2 Oo rD NA from the surface of SWCNTs by surfactants leads to astrong blue-shift and increase in the fluorescence intensity.I nterestingly,t he change in the fluorescence characteristics of pristine SWCNTs and DNA-coated SWCNTs immobilized in gel are highly similar. This suggests that considerable portions of the nanotube surface are exposed to H 2 O. [175] However,t odate, al ocal model of solvatochromism that accounts for the nonhomogeneous structure around SWCNTs is missing.
One of the best understood systems is the recognition of small molecules by DNA-functionalized SWCNTs.Here,the mechanism of sensors for catecholamine neurotransmitters such as dopamine have been studied in greater detail (Figure 19 d,e).
As these molecules are redox-active,they could reduce or oxidize either the SWCNT or the surrounding organic phase, thereby affecting the fluorescent properties.T os tudy this potential mechanism, the redox potential and the fluorescent response of certain analytes were correlated. This study showed that molecules with an egative redox potential are more likely to increase the SWCNT fluorescence (Figure 19 b). [63] However,a smolecules of the same redox potential can induce drastically different fluorescence responses,t he redox potential alone cannot account for the fluorescence changes observed. [63] Likewise,f luorescent responses of ssDNA-functionalized SWCNTs to dopamine and riboflavin cannot be correlated to the amount of adsorbed nucleotides/DNAmolecules on the SWCNT surface (Figure 19 c), which suggests that more complex conformational changes are responsible for the change in the SWCNT fluorescence. [176] Molecular dynamics simulations showed that as tacking of dopamine with DNA-functionalized SWCNTs leads to interactions between the phosphate backbone of the DNAa sw ell as the hydroxy and amine groups of dopamine (Figure 19 d,e). [100] As ar esult of this interaction, the phosphate backbone moves toward the SWCNT surface and the electrostatic potential at the SWCNT surface changes (Figure 19 d,e). [100] It is also known that the diffusion coefficient of the excitons in surfactant-containing systems changes with the surfactant identity and, furthermore,c orrelates with the fluorescence intensity. [48] As pristine SWCNTs do not show any fluorescence response to dopamine [100] and differently functionalized SWCNTs display different affinities to neurotransmitters, [118] it follows that the organic phase (DNA) governs both the sensitivity as well as selectivity for this neurotransmitter. [118] Theu sed biopolymers are typically charged and, consequently,e lectrostatic interactions play an integral part for biosensing. [177] It has been shown that the presence of certain salts alters the conformation of the DNAw rapping on the SWCNT [56,57] and decreases the electrostatic repulsions between equally charged molecules. [177] To reduce ioninduced fluorescence effects,t he flexibility of the DNAc an be altered by using xeno nucleic acids. [57] Agood example of the influence of electrostatic repulsion and screening effects is the increased SWCNT surface accessibility at higher salt concentrations (Figure 19 f). [177] In as urfactant-containing system, the exposed surface is covered by the surfactant, which causes ab lue-shift of the NIR fluorescence,a gain demonstrating that the dynamics of the organic phase around the SWCNT is crucial for the sensing mechanism. [177] By modulating the exposed surface area, it is furthermore possible to tune surfactant-suspended SWCNTs to respond to different bio-analytes. [137] Together, these findings indicate that fluorescence is modulated by the precise 3D arrangement of the molecules,i ons,a nd water molecules in the vicinity of the nanotube.

Exciton Decay and Defects
As described above,the introduction of certain sp 3 defects into the carbon lattice of SWCNTs can increase the NIR fluorescence through the trapping of excitons. [53,74] The defined introduction of quantum defects,t hus,p rovides away to perturb the exciton decay and elucidate the involved processes that ultimately affect the fluorescent response.A s the NIR fluorescent response of SWCNT-based sensors to dopamine in H 2 Oa nd D 2 Od id not show major differences, electronic-to-vibrational energy transfer (EVET) [178] seems not to be amain factor in (dopamine) sensing. [87] In contrast, the correlation between the length of the SWCNTs and their fluorescent response seems to indicate that quenching at the ends plays ar ole.H owever,t he fluorescence response was independent of the variation of defect density. [87] Together with the finding that as mall number of quantum defects reverse the fluorescent response of DNA-functionalized SWCNTs to dopamine (Figure 6d), it follows that multiple rate constants are affected by the analyte. [87] Computationally, the experiments were best explained by athree rate constant model (3RC) that includes ad ecrease in the nonradiative decay from the E 11 state (k nr ), an increase in the exciton diffusion constant (D e ), and an increase in the nonradiative decay constant from the E 11 * (k nr * )c aused by dopamine. [87] Together,t hese insights highlight the complex interplay between photophysics and molecular recognition as well as new avenues to tailor sensing using defects. Figure 19. Insights into the mechanism of SWCNT-baseds ensors.a )Change in the fluorescence signal of an individual (7,6)-SWCNTupon successivea dditiono fa cid (!)a nd base (~). Luminescence recovery is observed after base addition, which indicates direct quenchingby protons. Adapted from Ref. [47] with permission.b )Correlation between the fluorescence response of different SWCNT/polymer conjugates for different analytes( replicates ymbols), such as dopamine and ascorbic acid, and the redox potential. The spread along the y axis indicates that the redox potential alone cannot explain the fluorescence change. Adapted from Ref. [63] with permission.C opyright2 016 American Chemical Society. c) There is also no simple correlation between the fluorescence response and surface coverage, shown here for ssDNA-functionalizedS WCNTs and the response to riboflavin. Adapted from Ref. [176] with permission.C opyright 2019 American Chemical Society.d)Mode of interaction between dopaminea nd DNA-functionalized SWCNTs. The hydroxy groups of dopamine interact with the phosphate groups of the DNA backbone, which pulls them closer to the SWCNT surface. Most likely,s olvatione ffects play an important role. e) Corresponding potentiall andscape on aSWCNT functionalized with (GT) 15 DNA. The blue regions with changed potential colocalize with the dopaminei nteractions site and affect the exciton fate. In this particular case, the fluorescence quantum yield increases. Adapted from Ref. [100] with permission. f) Wavelength shift in response to an interaction between ssDNA-suspended SWCNTswith SDBS, asurfactant. The hybridization of ssDNA is influenced by the salt concentration and changes the surface accessibility of the SWCNT.Reprinted from Ref. [177] with permission.C opyright 2018 American Chemical Society.

Considerations on Kinetics and Imaging
Theprocesses related to sensing happen on acertain time scale.How fast an analyte binds or dissociates from asensor is determined by its kinetics.A dditionally,t he optical signal is detected in setups that determine aspects such as spatial resolution or imaging speed. In the following sections we discuss how these hallmarks of fluorescence sensors affect their performance.

Kinetics of Sensors and Impact on Spatiotemporal Resolution
Chemical imaging with many SWCNT-based sensors at one time is ah ighly effective strategy to gain chemical information from as ample with outstanding spatial and temporal resolution. [179] To understand how the collective image of such an array of sensors reflects the concentration of an analyte,M eyer et al. used stochastic Monte Carlo simulations to study the kinetic requirements for spatiotemporal chemical imaging with nanoscale sensors such as SWCNTs. [180] Thes ubject of the simulation was an anosensor array being exposed to ac hanging concentration gradient of an analyte.T op redict the image one gains from many sensors, single sensor responses were first simulated. In at ypical scenario,t he time-dependent concentration/diffusion profile of ad ynamic process,s uch as release of signaling molecules (e.g. neurotransmitters) from cells and the stochastic binding site state of the sensors for certain rate constants,w as simulated to calculate the expected fluorescence change of single sensors (Figure 20 a,b). Theo verall image was then calculated considering the individual fluorescence emission point spread functions and technical considerations,s uch as the frame rate of the camera. This simulation allows the prediction of how the rate constants of asensor (k on , k off )and other factors affect the spatiotemporal resolution, for example,t or esolve fast concentration changes such as neurotransmitter release from cells.Itcan serve as guiding principle for the chemical design but also for the interpretation of signals from ag iven biological problem. Phase diagrams (Figure 20 c) indicated that the senors need asurprisingly low affinity (K d = 100 mm)toresolve fast processes.

Ratiometric Detection
So far, optical biosensors based on SWCNTs have mainly been fabricated from mixtures of multiple chiralities.T his leads to alarge spectral overlap in the fluorescence emission, which complicates multiplexing and reduces sensitivity.
Advances in the separation and functionalization of SWCNTs have recently enabled the sole use of SWCNTs emitting below 1000 nm together with low-cost silicon-based detectors [145,181] for the development of ratiometric sensors, [20,29,30,32,33,112,121,154,169] in which two or more distinct NIR signals are detected simultaneously.H ere,o ne SWCNT chirality is typically not responsive to the analyte and acts as areference.Besides the fact that single chirality SWCNTs lead to six-to tenfold higher fluorescence intensities compared to multichirality SWCNT mixtures at the same concentration, [49,182] ratiometric approaches are more stable to external noise.D espite the clear advantages,t he implementation was not possible for al ong time because of difficulties in gaining chirality-pure SWCNTs.
Although progress was made in the synthesis of chiralityenriched SWCNT samples, [183] only af ew chirality-enriched samples are commercially available.Different separation and purification methods have been developed. They range from density gradient centrifugation, [184] gel chromatography, [185] ion-exchange chromatography, [55] and aqueous two-phase extraction [186] to wrappings of special macromolecules that preferably solubilize certain chiralities. [22,187] What they all have in common is that the resulting pure chiralities are solubilized in certain polymers or surfactants.H owever,f or ab iosensing application it is necessary to tailor the surface chemistry.T herefore,s traightforward processes are required that yield chirality-pure SWCNTs with tunable functionalization. [121] Thef irst ratiometric SWCNT-based sensor was demonstrated by Giraldo et al.,w ho integrated into the leaves of plants two sensors:one for the detection of H 2 O 2 and one for Figure 20. Kinetic requirements of sensors for spatiotemporal imaging. a) Astochastic-kinetic simulation allows the binding states (blue) to be predicted and consequently the fluorescence traces of single sensors in response to achanging concentration profile (red), for example, release of molecules from acell. b) Relative intensity response of asingle sensor over time based on the concentration profile shown in (a) and the assumption of 10 binding sites. c) Spatial and temporal resolution phase diagrama safunction of rate constants. The red regions show combinationsofrate constants that allow the detection of the biological events (here, release of molecules from acell). Reprinted from Ref. [180] with permission.C opyright 2017 American Chemical Society.
NO. [20] Thes ensor system was based on the ratio of the distinct emission bands of two chirality species (Figure 21 a). Whereas the (GT) 15 -(7,6)-SWCNT-based sensor was quenched by 20 %w ithin 10 min in the presence of 100 mm H 2 O 2 ,t he reference sensor remained mostly invariant to the analyte.T he overall sensor response was similar to in vitro tests.
Most recently,N ißler et al. combined the isolation of specific SWCNTs and their subsequent functionalization for the detection of neurotransmitters and other small molecules. [49] They used aqueous two-phase extraction to obtain chirality-pure (6,5)-, (7,5)-, (9,4)-, and (7,6)-SWCNTs and applied as urface exchange through dialysis to remove the DOC wrapping of the SWCNTs and replace it with specific ssDNAs equences or aptamers.T his approach enabled the fabrication of ratiometric sensors,f or example,f or the detection of dopamine and H 2 O 2 (Figure 22 b,c). Fort hese sensors,S WCNTs were functionalized either with a( GT) 40 sequence or, in the case of H 2 O 2 detection, with an aptamer (hemin binding aptamer,H eApta) that binds the protoporphyrin hemin and catalyzes the decomposition of H 2 O 2 .PEG-PL-functionalized SWCNTs of another chirality served as an invariant reference.T hese examples show the potential of ratiometric or multiplexed sensing and given the large wavelength range of SWCNT fluorescence,t here are plenty of opportunities for advanced sensing schemes.

Remote Imaging and Alternative Excitation Pathways
Remote imaging-the spatial separation of molecular sensors and detectors-is particularly beneficial to observe biochemical processes non-invasively,for example,inbiomanufacturing or in vivo.Asonly asmall portion of the emitted light is captured by acamera within acertain distance to the sensors,remote detection requires either bright fluorophores or cameras with ahigh quantum yield in the spectral window of the fluorophore emission.
Additionally,t his setup was modified for SWCNT detection by using an NIR-sensitive InGaAs camera, awhite-light LED,a nd corresponding filters for wavelength-specific excitation and detection of emission (Figure 22 a). Up to now, remote imaging of fluorescent SWCNT-based sensors has been used for the identification of bacteria [29] and monitoring of plant health, [30][31][32]148] for which resolutions in the (sub-)millimeter range were achieved with standoff distances of up to 1m.
Apart from exploiting the decreasing sensitivity of Sidetectors in the NIR, efforts have been made to promote NIR-fluorescent transducers using straightforward designs for inexpensive NIR fluorimeters. [167] Instead of using high-cost InGaAs photodiode arrays,s ingle InGaAs diodes were combined with am otorized stage controlled with an open source programming language.T he robustness of these devices outside of the laboratory was demonstrated in ah igh-throughput format with field-side measurements of soil samples.F uture developments might also show remote detection using pure (6,4)-SWCNTs with Si-based cameras and also versatile use as smart surfaces,f or example,f or monitoring contamination with pathogens,s uch as bacteria, on medical surfaces or even implants.
Fluorophore brightness is not the only performancerelated factor when measurements in live biological tissues are carried out. Fluorophore stability as well as wavelengthdependent tissue scattering,a bsorption, and the background originating from autofluoresence must also be considered. From this point of view,S WCNTs have excellent photophysical properties.
However,i maging at the single molecule level is always accompanied by considerations to achieve ah igh signal to noise ratio.L ong-term single SWCNT imaging has,i n particular,b ecome established for (6,5)-SWCNTs in living cells [191] and brain tissue. [192] Although the second-order excitonic transition, E 22 (S 22 ), is typically used for excitation, alternative excitation pathways such as the K-momentum exciton-phonon sideband (KSB) excitation [193] and up-conversion excitation [194] have been successfully demonstrated, but excitation efficiency, photostability,a sw ell as absorption and scattering of molecules in tissue differ depending on the excitation wavelength. DannØ et al. evaluated the different excitation options (Figure 22 b) for optimal single (6,5)-SWCNT imaging. [189] Here,E 22 excitation of PEG-PL SWCNTs was found to be four times more efficient than KSB excitation and an order of magnitude more efficient than 1064 nm up-conversion excitation, while the signal to noise ratio was more than five times higher for KSB and up-conversion excitation. However,t he excitation at the E 22 transition is not ideal due to limited tissue penetration depth and autofluorescence.I na ddition, simulations to quantify the impact of tissue absorption showed that ah igher temperature rise of the tissue induced by upconversion excitation might be an issue,t hus suggesting that KSB excitation is the best choice when considering all the factors.Nevertheless,itstill requires relatively high excitation doses in the kW cm À2 regime.
Recently,single SWCNT imaging was performed in brain tissue in vivo by using ultralow excitation doses of 0.1 kW cm À2 (Figure 22 c). [190] To achieve this,s p 3 defects were introduced into (6,5)-SWCNTs,which lead to afluorescence emission at E 11 * (1160 nm) when exciting at the firstorder excitonic transition E 11 (985 nm). This approach is beneficial as aresult of excitation in the NIR window,but also because of the increased brightness by channeling free excitons to defect sites and subsequent E 11 * emission. Another way to both excite and detect in the NIR region is the use of multiphoton microscopy.This technique relies on the nonlinear excitation of at least two photons and is optimal for in vivo tissue applications when using NIR radiation. Del Bonis-O'Donnell et al. demonstrated two-photon 1560 nm excitation of dopamine-sensitive SWCNT-based sensors, which showed only 4% scattering (one-photon excitation: 42 %s cattering). [195] However,t he frame rate is significantly reduced for single sensor imaging compared to wide-field one-photon excitation. [196]

Hyperspectral and Spinning Disc Microscopy
Hyperspectral microscopy (i. e. simultaneous imaging at different wavelengths) provides another approach to exploit the spectral variety for multiplexed SWCNT imaging. Roxbury et al. resolved up to 17 distinct chiralities with single nanotube spatial resolution. [197] In contrast to organic fluorophores,m ore chiralities can be imaged simultaneously in acertain emission window due to the narrow emission bands of SWCNTs.The hyperspectral imaging is based on the use of avolume Bragg grating (VBG) placed between the emission port of an inverted fluorescence microscope and the NIR camera. TheV BG filters one specific emission wavelength depending on optical properties such as the incident angle q and the grating period L (Figure 23 a).
Thespatial imaging of different chiralities is then achieved by measuring acontinuous stack of 152 images within atime frame of 20 sa nd 10 min depending on the signal intensity and, thus,t he integration time.B yu sing this approach 12 different chiralities from DOC-SWCNTs in live human cervical cancer cells could be detected (Figure 23 b). Additionally,i ndividual SWCNTs adsorbed on as urface in live mammalian cells,m urine tissues ex vivo,a nd zebrafish endothelium in vivo were imaged.
Another challenge of typical wide-field microscopy setups is the poor z-resolution. To improve this limitation, aN IR spinning-disc confocal laser microscope with an increased resolution and imaging contrast was demonstrated by Zubkovs et al. (Figure 23 c). [198] Thecustom-built microscope was based on aspinning-disc module integrated between acooled InGaAs camera and the microscope body,which rejects out of focus light.
To achieve amaximized photon intensity,the lenses in the spinning disc unit were optimized for the NIR region. The authors showed in different biological applications the advantages of an improved lateral/axial resolution of 0.5 AE 0.1 mm/0.6 AE 0.1 mm( enhancement of 17 %/45 %) compared to the wide-field configuration, reaching from single-particle tracking over the spatial distribution of nanoparticles within Angewandte Chemie Reviews an organelle to the optical in situ monitoring of glucose by GOx-SWCNT-based sensors embedded within an agarose gel. Overall, this approach showcases the opportunities for in vitro and in vivo imaging and sensing with improved spatiotemporal resolution.

Outlook and Perspectives
SWCNTs have shown their large potential as versatile building block for biosensors.T he last few years have provided fundamental mechanistic insights,n ovel recations, and novel applications such as sensing in plants or primary cells.T he advent of covalent quantum defect chemistry as well as the broader availability of monochiral samples will further advance typical figures of merit such as selectivity and robustness in demanding biochemical environments.O ne of the key challenges remains ab asic understanding of molecular recognition and also signal transduction in the organic phase around these materials.A dvances in this space can directly translate into superior selectivities.A dditionally, high-throughput or screening approaches will increase the speed of chemical discoveries.T his is particularly interesting for applications in complex biological environments,w here interactions become too complex to be predicted. Several milestones remain to be achieved before transfer into commercially available products.Invivo applications require long-term evaluation of each sensorstoxicology and stability profile.Here,new covalent quantum defect functionalization strategies as well as the standardization of protocols and the purity of SWCNTs offer promising opportunities.A dditionally,h igher degrees of multiplexing and the adaptation of conventional cameras and readout systems will increase the application potential of SWCNT-based sensors.I ns ummary, SWCNT-based biosensors offer arich playground for chemistry and related disciplines and promise further advances and breakthroughs in the near future. Figure 23. Advances in spatial and spectral resolution.a )AVolume Bragg grating filters one specific emission wavelength depending on the incident angle q,r efractive index n,and grating period L,which enables hyperspectral imaging. b) Detection of 12 different SWCNT chiralities without deconvolution in live human cervical cancer cells. Reprinted from Ref. [197] with permission.c)Improved NIR image contrast of NIR fluorescentbeads with aspinning disc NIR fluorescence microscopei ncomparison to wide-field microscopy. Reprinted from Ref. [198] with permission. Yes NO [128] In cultures of A375 melanoma cells through micropinocytosis, NO production using NO-releasing anticancer drug JS-K VEGF-mediated NO production in endothelial cells (AT) 15  [a] FI:f luorescence intensity,ND: not determined,* *: limitedbythe Abbe limit, diffusion, and detection speed.  [15] In PBS, immobilized Screening approach: